May 09, 2023

Public workspaceTS Spurrs - FFPE block - 1mm3 cube cut out - tissue (TM - 013)

DOI
dx.doi.org/10.17504/protocols.io.kxygx968wg8j/v1
  • 1CSIRO ACDP
sandra.crameri
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DOI: dx.doi.org/10.17504/protocols.io.kxygx968wg8j/v1
Protocol Citation: sandra.crameri 2023. TS Spurrs - FFPE block - 1mm3 cube cut out - tissue (TM - 013). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx968wg8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 09, 2023
Last Modified: May 09, 2023
Protocol Integer ID: 81632
Abstract
Processing unstained parraffin block that has had a 1mm3 piece disected out of the face to Spurr's resin block for EM.
Guidelines
All step times are the minimum time required. Longer steps are acceptable.
Protocol materials
Reagentxylene
In 3 steps
ReagentEthanol
Step 5
ReagentEthanol
In 2 steps
ReagentEthanol
Step 7
ReagentGlutaraldehyde aqueous solution 25% EM Grade ampoulesProSciTechCatalog #C002
Step 8
ReagentOsmium TetroxideProSciTech
Step 10
Reagentethanol
In 4 steps
HEADER
HEADER
SAN:


SPEC No:


OPERATOR & STEPS:


OPERATOR & STEPS:


Dewax FFPE sections
Dewax FFPE sections
15m
15m
Reagentxylene or X3B DurationOvernight at Temperature60 °C

5m
Reagentxylene or X3B Duration00:05:00 at TemperatureRoom temperature

5m
Reagentxylene or X3B Duration00:05:00 at TemperatureRoom temperature

5m
Rehydrate
Rehydrate
15m
15m
Concentration100 % volume ReagentEthanol Duration00:10:00

10m
Concentration95 % volume ReagentEthanol Duration00:10:00

10m
Concentration70 % volume ReagentEthanol Duration00:10:00

10m
Conventional - with reduced times for 5um thick FFPE section
Conventional - with reduced times for 5um thick FFPE section
2h 50m
2h 50m
Concentration2.5% % volume ReagentGlutaraldehyde aqueous solution 25% EM Grade ampoulesProSciTechCatalog #C002 in Concentration0.1 Molarity (M) Sorenson's phosphate buffer Ph07.2 300 mosmol/kgDuration00:20:00 .

20m
Wash buffer Duration00:05:00

5m

Concentration1 Mass / % volume ReagentOsmium TetroxideProSciTech in bufferDuration01:00:00

1h
Concentration70 % volume ReagentEthanol Duration00:10:00

10m
Concentration95 % volume Reagentethanol Duration00:10:00

10m
Concentration100 % volume Reagentethanol Duration00:10:00

10m
Concentration100 % volume dry Reagentethanol Duration00:10:00

10m
Concentration100 % volume Spurrs:Concentration100 % volume dry Reagentethanol in 1:1 ratioDuration00:30:00

30m
Concentration100 % volume SpurrsDuration01:00:00

1h
Concentration100 % volume SpurrsDuration01:00:00

1h
Oven polymerise Temperature65 °C DurationOvernight